A wide variety of viral conditions affect living organisms. Many viral conditions affect the majority of humans, or other mammals, at some time in their lives. Other viruses affect a smaller but significant number of humans at some time. Many of these viruses must fuse with a host cell membrane in order to infect the cell and reproduce. Enveloped viruses include a fusion protein that changes conformation from a native form to a fusogenic form. This promotes fusion of the viral membrane with the host cell membrane, resulting in injection of viral contents into the host cell.
Influenza pandemics recur on an annual basis worldwide. Vaccination programs aimed at curtailing the spread of the disease are hampered by the fast mutation rate of antigenic sites on the virus. Medical practices abroad permit use of the drugs amantadine and rimantadine to treat influenza A infections. However, due to the potential for undesirable side effects, use in the United States is recommended only for the population deemed most at risk. The cost of millions of lives and billions of dollars each winter underscores the urgent need for development of safe and effective anti-influenza drugs.
Analysis of the replication pathway of the orthomyxovirus reveals a number of steps that can be targeted for antiviral therapy. Successful infection requires host cell recognition, delivery of the infectious genome into the host cell cytoplasm, replication of the viral genes and proteins, and escape of progeny viruses. Any of these steps is potentially susceptible to intervention. However, the antiviral strategy must be specific for influenza proteins or processes in order to avoid adventitious inhibition of normal cellular functions.
Early events in the viral life cycle leading to the deposition of the viral genes inside the cell are shown schematically in FIG. 1. Referring to FIG. 1, infection begins by binding between the hemagglutinin glycoprotein 1 protruding from the viral envelope 2 and sialic acid residues of cellular receptors 3, which triggers endocytosis (at B). As the virus is endocytosed via the normal cellular pathway it encounters progressively decreasing pH. At a threshold pH specific to the particular strain of influenza, fusion between the viral membrane and the endosomal membrane is initiated (at C). This fusion event results in release of the infectious genome 5 into the cell cytoplasm 6 (at D), where successive steps of the replication cycle can occur. M. Kielian and S. Jungerwirth, "Mechanisms of Enveloped Virus Entry into Cells," Mol. Biol. Med. 7:17-31 (1990); Simons, K., Garoff, H. and Helenius, A., "How an animal virus gets into and out of its host cell," Sci. Am. 246: 58-66 (1982).
The critical role of membrane fusion in infection makes it an attractive target for inhibition. To date, this route of antiviral chemotherapy has been largely unexplored. Inhibition of fusion has the advantage of interfering with an early step in replication, prior to penetration of the virus into the host cell. As it does not aim to inhibit an enzymatic activity or to mimic any ligands, the chance of fortuitous inhibition of unintentional targets is minimized. Since fusion is a step common to the replication of all enveloped viruses, this antiviral strategy can potentially be applied to a host of other viral diseases, including those caused by togaviruses, rhabdoviruses, paramyxoviruses, herpes viruses, and retroviruses.
Examination of fusion in more detail reveals it to be a protein-mediated event triggered by the viral hemagglutinin. Hemagglutinin is a trimer of identical subunits. Each monomer is composed of two polypeptide chains, HA1 and HA2, which are generated by proteolytic cleavage of a precursor, HA0. The polypeptides comprising the monomer are covalently linked by a single disulfide bond but the three monomers of a trimer are stabilized by noncovalent interactions only. Wilson, I. A., Skehel, J. J. and Wiley, D. C., "Structure of the hemagglutinin membrane glycoprotein of influenza virus at 3 .ANG. resolution," Nature 289: 366-73 (1981). The chains in the trimer are sometimes described as the A, C and E chains (HA1 chains in each monomer) and B, D, and F chains (corresponding HA2 chains).
Residues 1-24 at the amino terminus of HA2 play a critical role in fusion. This segment, known as the fusion peptide, has been proposed to form a sided helix in which one face of the helix is composed primarily of hydrophobic amino acids. White, J. M., "Viral and cellular membrane fusion proteins," Ann. Rev. Physiol. 52: 675-97 (1990). While the function of the fusion peptide is not clearly understood, current evidence suggests it aids fusion by interacting with the target membrane. Stegmann, T., Delfino, J. M., Richards, F. M. and Helenius, A., "The HA2 subunit of influenza hemagglutinin inserts into the target membrane prior to fusion," J. Biol. Chem. 266: 18404-10 (1991). The fusion peptide is the most highly conserved region among influenza virus hemagglutinins sequenced to date, and hydrophobic or sided fusion peptide sequences have been identified in the fusion proteins of a wide variety of enveloped viruses. White, J. M., loc. cit.
Upon exposure to low pH, hemagglutinin undergoes an irreversible conformational change that is a prerequisite for fusion. The conformational change most likely involves a rearrangement of domains rather than major secondary structural alterations since circular dichroism measurements reveal only minor differences between the neutral and low pH forms. Studies of the conformational change have been facilitated by isolation of the soluble ectodomain of the integral membrane glycoprotein. This proteolytic fragment, BHA, is generated by bromelain cleavage of hemagglutinin at a site adjacent to the transmembrane domain. BHA has been identified as a reliable model for the complete protein (HA) in many assays not requiring membrane attachment. See White, J. M. and Wilson, I. A., "Anti-peptide antibodies detect steps in a protein conformational change: low pH activation of the influenza virus hemagglutinin," J. Cell Bio. 105: 2887-96 (1987). The term "hemagglutinin" is used to mean both HA, the intact integral membrane protein, and BHA, its proteolytic fragment lacking the transmembrane and cytoplasmic domains.
Previous studies on HA and BHA have shown that the low pH form of hemagglutinin may be distinguished from the neutral pH form immunologically, biochemically and biophysically. Only the low pH conformation is susceptible to cleavage by trypsin and by proteinase K. The low pH form of BHA has increased hydrophobic character, observed by binding to liposomes, partitioning into detergent solution or aggregation in aqueous solution. Low pH and native hemagglutinin have also been distinguished by electron microscopy.
The crystal structure of neutral pH BHA from the X31 strain of influenza (A/Hong Kong/1968; H3N2) has been solved to 3 .ANG. resolution. Wilson, I. A., et al., loc. cit. Referring to FIG. 2, tracings of the .alpha.-carbon backbones of both the trimer (left) and monomer (right) are shown. The figure illustrates the bromelain cleavage site 11, the C-terminus 13 of chain HA1, and fusion peptide between 14 and 15. Each monomer has been described as comprising three domains: a globular head domain 12 containing the sialic acid binding site, a narrow stem composed primarily of residues of HA2, and a connecting hinge region. The fusion peptides of native hemagglutinin are buried in the trimer interface of the stem region. The conformational change is thought to release the fusion peptides from their unexposed location, freeing them to mediate fusion.
Since membrane fusion depends on the conformational change, inhibition of fusion peptide exposure should prevent fusion and all successive steps of viral replication. Therefore, the antiviral strategy was to identify a small molecule that could bind to the native form of hemagglutinin and stabilize that conformation over any fusogenic conformation. A new class of such inhibitors may provide safe and effective drugs.